Transcutaneous auricular vagus nerve stimulation therapy in patients with cognitively preserved structural focal epilepsy: A case series report.

OBJECTIVE: Transcutaneous auricular vagus nerve stimulation (taVNS) was performed in two patients suffering structural focal epilepsy with preserved intellectual ability to show the feasibility of taVNS for specific patient groups. CASE PRESENTATIONS: Patient 1 was a 24-year-old woman with frontal lobe epilepsy who had weekly hyperkinetic seizures despite multiple anti-seizure medications. Patient 2 was a 27-year-old woman with parietal lobe epilepsy and focal cortical dysplasia in the vicinity of the lipoma in the corpus callosum. She experienced weekly focal-impaired awareness seizures even with anti-seizure medication. taVNS was applied to the left earlobe of both patients at 1.5 mA, 25 Hz, 250 µs pulse width, and 30 s stimulation with 30 s rest for 4 h per day. Over an 8-week baseline and 20 weeks of stimulation, the rate of reduction in seizure frequency was evaluated, along with quality-of-life using the Short-Form 36-Item Health survey. RESULTS: At baseline, we measured up to 11 and 12 focal seizures per week in Patient 1 and 2, respectively, with both patients achieving seizure freedom after 4 and 20 weeks taVNS, respectively. Patient 1 and 2 were observed for 18 and 14 months, respectively, including the clinical trial and follow-up observation period. Quality-of-life ratings increased in both patients, and no significant adverse events occurred during the study period. During the maintenance period after 20 weeks, seizures remained absent in Patient 1, and seizures remained reduced in Patient 2. CONCLUSION: Our results demonstrate that taVNS may be a promising tool for structural focal epilepsy with preserved cognitive function. A multicenter double-blind clinical trial is needed to confirm the role of taVNS as an anti-seizure tool.

An integrated genetic analysis of epileptogenic brain malformed lesions.

Focal cortical dysplasia is the most common malformation during cortical development, sometimes excised by epilepsy surgery and often caused by somatic variants of the mTOR pathway genes. In this study, we performed a genetic analysis of epileptogenic brain malformed lesions from 64 patients with focal cortical dysplasia, hemimegalencephy, brain tumors, or hippocampal sclerosis. Targeted sequencing, whole-exome sequencing, and single nucleotide polymorphism microarray detected four germline and 35 somatic variants, comprising three copy number variants and 36 single nucleotide variants and indels in 37 patients. One of the somatic variants in focal cortical dysplasia type IIB was an in-frame deletion in MTOR, in which only gain-of-function missense variants have been reported. In focal cortical dysplasia type I, somatic variants of MAP2K1 and PTPN11 involved in the RAS/MAPK pathway were detected. The in-frame deletions of MTOR and MAP2K1 in this study resulted in the activation of the mTOR pathway in transiently transfected cells. In addition, the PTPN11 missense variant tended to elongate activation of the mTOR or RAS/MAPK pathway, depending on culture conditions. We demonstrate that epileptogenic brain malformed lesions except for focal cortical dysplasia type II arose from somatic variants of diverse genes but were eventually linked to the mTOR pathway.

Efficacy of sirolimus for epileptic seizures in childhood associated with focal cortical dysplasia type II.

OBJECTIVE: The efficacy of the mechanistic target of rapamycin inhibitor, sirolimus, was recently reported for patients more than 6 years of age by Kato et al. We evaluated the efficacy and safety of sirolimus in a 2-year-old patient with recurrent focal seizures with impaired consciousness after focal cortical dysplasia (FCD) type IIa resection. METHODS: The patient was a 2-year-old girl who had recurrent seizures after undergoing FCD resection at 4 months of age. The initial dose of sirolimus was 0.5 mg/day and was gradually increased using the trough blood concentration before oral administration as an index, and evaluation was performed at 92 weeks. RESULTS: The trough blood level of sirolimus was increased to 6.1 ng/mL and maintenance therapy was started at 40 weeks. Focal seizures with impairment of consciousness with tonic extension of the limbs decreased. No critically serious adverse events occurred. CONCLUSION: Sirolimus was effective against epileptic seizures from FCD type II even for a child under 5 years of age. There were no critically serious adverse events and administration could be continued.

Reinterpretation of magnetic resonance imaging findings with magnetoencephalography can improve the accuracy of detecting epileptogenic cortical lesions.

OBJECTIVE: This study examined whether the application of magnetoencephalography (MEG) to interpret magnetic resonance imaging (MRI) findings can aid the diagnosis of intractable epilepsy caused by organic brain lesions. METHODS: This study included 51 patients with epilepsy who had MEG clusters but whose initial MRI findings were interpreted as being negative for organic lesions. Three board-certified radiologists reinterpreted the MRI findings, utilizing the MEG findings as a guide. The degree to which the reinterpretation of the imaging results identified an organic lesion was rated on a 5-point scale. RESULTS: Reinterpretation of the MRI data with MEG guidance helped detect an abnormality by at least one radiologist in 18 of the 51 patients (35.2%) with symptomatic localization-related epilepsy. A surgery was performed in 7 of the 51 patients, and histopathological analysis results identified focal cortical dysplasia in 5 patients (Ia: 1, IIa: 2, unknown: 2), hippocampal sclerosis in 1 patient, and dysplastic neurons/gliosis in 1 patient. CONCLUSIONS: The results of this study highlight the potential diagnostic applications of MEG to detect organic epileptogenic lesions, particularly when radiological visualization is difficult with MRI alone.

Efficacy of bezafibrate for preventing myopathic attacks in patients with very long-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a mitochondrial fatty acid oxidation disorder that causes episodic attacks, such as general fatigue, hypotonia, myalgia, and rhabdomyolysis accompanied by lack of energy. As yet, there are no preventative drugs for these VLCADD-associated metabolic attacks. PATIENTS AND METHODS: We conducted an open-label, non-randomized, multi-center study into the effects of bezafibrate on five patients with VLCADD. Bezafibrate was administered for 4 years, and we analyzed the number of myopathic attacks requiring hospitalization and treatment infusions. RESULTS: The number of myopathic attacks requiring infusions of 24 h or longer significantly decreased during the study period. The patients' ability to conduct everyday activities was also improved by the treatment. CONCLUSION: Our findings show the potential long-term efficacy of bezafibrate in preventing myopathic attacks for patients with VLCADD.

Long-term follow up of an adult with alternating hemiplegia of childhood and a p.Gly755Ser mutation in the ATP1A3 gene.

Alternating hemiplegia of childhood (AHC) is a rare neurological disease mainly caused by mutations in the ATP1A3 gene and showing varied clinical severity according to genotype. Patients with a p.Gly755Ser (p.G755S) mutation, one of minor genotypes for AHC, were recently described as having a mild phenotype, although their long-term outcomes are still unclear due to the lack of long-term follow up. Here, we demonstrate the full clinical course of a 43-year-old female AHC patient with p.G755S mutation. Although her motor dysfunction had been relatively mild into her 30 s, she showed a subsequent severe aggravation of symptoms that left her bedridden, concomitant with a recent recurrence of seizure status. The seizures were refractory to anti-epileptic drugs, but administration of flunarizine improved seizures and the paralysis. Our case suggests that the phenotype of AHC with p.G755S mutation is not necessarily mild, despite such a presentation during the patient's younger years.

The presence of short and sharp MEG spikes implies focal cortical dysplasia.

PURPOSE: This study focused on the characteristic needle-like epileptic spikes of short duration and steep shape seen on magnetoencephalography (MEG) in patients diagnosed with focal cortical dysplasia (FCD) morphologically. We aimed to validate the analysis of MEG spike morphology as a noninvasive method of identifying the presence and location of FCD. METHODS: MEG was collected by 204-channel helmet-shaped gradiometers. We analyzed MEG spike sources for 282 patients with symptomatic localization-related epilepsy. MEG showed clustered equivalent current dipoles when superimposed on their three-dimensional-magnetic resonance images (MRI) in 85 patients. Fifty-seven patients were excluded from our study, because they had destructive brain lesions or an insufficient number of spikes for statistical analysis. Twenty-eight patients (18 males, 10 females; aged 1-34 years) were finally matched to our inclusion criteria, and were categorized into three groups: FCD (7 patients), non-FCD (10 patients), and non-lesion (11 patients), based on the MRI findings. We measured the duration, amplitude, and tilt manually for at least 15 spikes per patient, and compared the three groups using a one-way analysis of variance, followed by the Tukey test when statistically significant (p < 0.05). In 17 patients with visible MRI lesions, we investigated the correlation between the depth of the lesion and the tilt using the Pearson product moment correlation. RESULTS: The average spike duration was significantly shorter in the FCD and non-lesion groups than in the non-FCD group (p < 0.05). The average amplitude was not significantly different between the three groups. The average spike tilt was significantly steeper in the FCD group than in the non-FCD group (p = 0.0058). There was no significant difference between FCD and non-lesion patients in both duration and tilt. Our additional study revealed a significant negative correlation between the depth of the lesion and the average tilt (p = 0.0009). SIGNIFICANCE: MEG epileptiform discharges of short duration and steep tilt characterize FCD, especially when located at the superficial neocortical gyrus. We speculate that this particular spike morphology results from the intrinsic epileptogenicity of FCD. Morphological analysis of MEG spikes can evaluate the etiology of epileptogenic lesions and detect a strong, localized epileptogenic focus such as that typically observed in FCD.

Induction of matrix metalloproteinase gene expression in an endothelial cell line by direct interaction with malignant cells.

Mouse endothelial TKD2 cells in monolayers were cocultured with various human cell lines for 24 h, and the expression of several secreted matrix metalloproteinases (MMP) and cell adhesion molecules was examined by real-time reverse transcription-polymerase chain reaction using mouse-specific primers. Coculture with normal fibroblasts did not elicit the expression of these molecules, but coculture with cancer cells induced the expression of MMP-3, MMP-9 and MMP-10 mRNA in endothelial cells, and in normal mouse embryonic fibroblasts. The induction of MMP mRNA was dependent on direct cell adhesion, as separate culture of A549 cells in Boyden chambers did not induce MMP mRNA, and neutralizing antibody against VLA-4 abolished the induction. An inhibitor of phosphatidylinositol-3-phosphate kinase strongly suppressed the induction of MMP-3, MMP-9 and MMP-10 mRNA, and expression of the dominant-negative mutant of phosphatidylinositol-3-phosphate kinase also decreased the induction. It was suggested that intracellular reactive oxygen species (ROS) levels were increased in TKD2 cells following adhesion to cancer cells. ROS scavengers decreased the levels of MMP induction, and roterone, an inhibitor of mitochondrial complex I, strongly suppressed the induction of MMP-3, MMP-9 and MMP-10. The depletion of mitochondria in TKD2 cells decreased the induction of MMP-9, but the induction of MMP-3 and MMP-10 was not affected. These results indicate that the adhesion of cancer cells to endothelial cells activates several distinct signaling pathways to induce MMP gene expression, and the pathways for MMP-3, MMP-9 and MMP-10 are partly different. For the induction of MMP-9, mitochondria participate in induction, possibly through the production of ROS.

Anthracyclines, small-molecule inhibitors of hypoxia-inducible factor-1 alpha activation.

Hypoxia-inducible factor-1 (HIF-1) is a central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. To identify small molecule inhibitors of the HIF-1 transcriptional activation, we have established a high through-put assay system using a stable transformant of mammalian cells that express the luciferase reporter gene construct containing a HIF-1 binding site. Using this system, we screened 5000 cultured broths of microorganisms, and we found that cinerubin (1-hydroxy aclacinomycin B) showed a significant inhibition of the reporter activity induced by hypoxic conditions. In addition, we demonstrated that aclarubicin also inhibited the HIF-1 transcriptional activity under hypoxic conditions, but neither doxorubicin nor daunorubicin inhibited it. Consistent with these results, cinerubin and aclarubicin inhibited the hypoxic induction of the vascular endothelial growth factor (VEGF) protein in HepG2 cells, but neither doxorubicin nor daunorubicin affected it. Thus, our results suggested that some anthracyclines are also acting as angiogenesis inhibitors.

Inhibition of angiogenesis and HIF-1alpha activity by antimycin A1.

We identified antimycin A1 as an inhibitor of the hypoxia-response element (HRE) from screening using a reporter under the control of HRE under hypoxic conditions. Antimycin A1 was effective at 20 pg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by antimycin A1. Angiogenesis induced by implantation of mouse sarcoma-180 cells was significantly inhibited by non-toxic doses of antimycin A1. Hypoxia inducible factor (HIF)-1alpha protein levels were significantly decreased by antimycin A1, but its mRNA level was not affected. Antimycin A1 is known to be an inhibitor of mitochondrial electron transport system, and depletion of mitochondria abolished antimycin A1-effect, at least in part. Inhibitors of proteasome or protein synthesis did not affect the decrease in HIF-1alpha level induced by antimycin A1. These results indicate that antimycin A1 inhibited angiogenesis through decrease in VEGF production caused by inhibition of HIF-1alpha activation.

Dynamic statistical parametric mapping for analyzing the magnetoencephalographic epileptiform activity in patients with epilepsy.

Our current purpose is to evaluate the applicability of dynamic statistical parametric mapping, a novel method for localizing epileptiform activity recorded with magnetoencephalography in patients with epilepsy. We report four pediatric patients with focal epilepsies. Magnetoencephalographic data were collected with a 306-channel whole-head helmet-shaped sensor array. We calculated equivalent current dipoles and dynamic statistical parametric mapping movies of the interictal epileptiform discharges that were based in the minimum-L2 norm estimate, minimizing the square sum of the dipole element amplitudes. The dynamic statistical parametric mapping analysis of interictal epileptiform discharges can demonstrate the rapid change and propagation of interical epileptiform discharges. According to these findings, specific epileptogenic lesion-focal cortical dysplasia could be found and patients could be operated on successfully. The presurgical analysis of interictal epileptiform discharges using dynamic statistical parametric mapping seems to be promising in patients with a possible underlying focal cortical dysplasia and might help to guide the placement of invasive electrodes.

Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids.

Screening using a reporter under the control of the hypoxia-response element (HRE) identified several flavonoids and homoisoflavonoids that inhibit the activation of HRE under hypoxic conditions. Among various compounds, isorhamnetin, luteolin, quercetin, and methyl ophiopogonanone B (MOB) were effective at 3 to 9 microg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by MOB in HepG2 cells, but the effective doses were 10 to 20 microg/ml. MOB caused destabilization of hypoxia-inducible factor (HIF)-1alpha, as revealed by Western blotting, that was dependent on proteasome activity and the tumor suppressor, p53. The tubular formation and migration of human umbilical vein endothelial cells was also inhibited by MOB. MOB is expected to act as an inhibitor of angiogenesis.

HIF-1-dependent VEGF reporter gene assay by a stable transformant of CHO cells.

For the establishment of a screening system to detect inhibitors of vascular endotherial growth factor (VEGF) expression, a stable transformant of Chinese hamster ovary cells was isolated and cloned by transfection of a hypoxia-inducible factor 1 (HIF-1)-dependent VEGF promoter reporter gene. The expression of the reporter gene in the clone cells, as measured by luciferase activity, was stable. Hypoxic responses were best observed at an initial cell density of 2 x 10(4)/well. The maximal increase of luciferase activity was 30 fold. In the highest cell density of 8 x 10(4)/well (2.1 x 10(5)/cm(2)), basal activity was increased 13-15 fold compared to that at the lower cell densities, and did not respond to hypoxia. Addition of CoCl(2), which is known to mimic hypoxia, increased luciferase activity more than 10 times in normoxia. Nitric oxide donors, which are known to suppress the activation of HIF-1, inhibited expression of the VEGF promoter reporter gene under hypoxia. Histone deacetylase inhibitors, trichostatin A and sodium n-butyrate which are known to stimulate transcription of many genes enhanced its transcription in hypoxia. These results indicate that the stable transformant is a useful tool for screening of HIF-1 modifiers.

Induction of p16/INK4a gene expression and cellular senescence by toyocamycin.

We constructed an assay system of a luciferase reporter with p16/lNK4a gene transcriptional regulatory domain to identify p16-inducing substances, and found toyocamycin to induce gene expression from the screening of culture fluids of Streptomyces. Toyocamycin is a nucleoside analog, and it increased the p16 mRNA level in human normal fibroblasts or synovial cells as assessed by Northern blot hybridization or real time RT-PCR. It also induced cellular senescence in normal human fibroblasts. The transcriptional regulatory regions of human p16 gene that were responsible for the induction were analyzed using deletion mutants of the transcriptional regulatory region of p16 linked to the luciferase gene. The DNA fragment -111 to +1 bp from the cap site was sufficient to drive toyocamycin-activated transcription of p16/luciferase reporter. Nucleotide sequences within this domain contained the Sp1- and Ets-binding sequences. Mutations were introduced into these sequences, and the Sp1 sequence was found to be critical for the induction, and this notion was confirmed from gel-mobility shift assay.

Induction of p16INK4a transcription and of cellular senescence by aclacinomycin-derivatives and cardiac glycosides.

Stable transformants of Saos-2 cells that contain the luciferase reporter gene under the control of the human p16INK4a transcriptional regulatory region were established, and were used to identify growth-inhibiting substances from culture broths of actinomycetes and extracts of plants. Among the active substances so far identified were an aclacinomycin-derivative, cenerubin B, and a cardiac glycoside, periplocin. These substances inhibited growth of normal human fibroblasts, and induced senescent phenotypes including a flattened morphology and increased acidic beta-galactosidase activity, although the activities of their derivatives to induce p16CDKN2 and beta-galactosidase did not coincided with each other. These results suggest that the reporter system using the p16CDKN2 transcriptional regulatory region is a useful means for screening growth inhibiting substances that are potential anti-tumor agents.